Hepatitis C virus (HCV) is an important worldwide cause of liver disease. Current standard of care (SOC) agents are inadequate for most patients, and new virus-specific drugs are urgently needed. Our long-term objective is to bring a new class of anti-HCV drugs to the clinic. We discovered that the HCV NS4B protein specifically binds a key segment of the HCV RNA genome, genetically validated the importance of this interaction for HCV replication, and used a microfluidics screen to identify clemizole-a first generation HI antihistamine widely used in the 1950s and 60s--as a potent fortuitous specific inhibitor of NS4B RNA binding. We have now successfully completed the milestones associated with the 3 specific aims of our Phase I ap- plication and now hypothesize: 1) clemizole is fully compatible with other drugs used, or soon to be used, for treating HCV; 2) clemizole's in vivo resistance profile can be precisely characterized and is predictable using a humanized mouse model; 3) clemizole has dramatic in vivo synergy with protease inhibitors (PIs) in advanced clinical development; 4) ICH criteria-compatible safety and toxicity studies will support advanced clinical development of clemizole. We seek to test these hypotheses, and further advance clemizole to an IND filing and successful commercialization, by performing our specific aims and associated milestones, which are to: Aim 1-Analyze key drug-drug interactions with expected cocktail components by determining: a) clemizole's PK and metabolite profiles in patient-derived samples and the reciprocal effect of SOC agents b) reciprocal effects of clemizole and PIs on each other's PK profile in the humanized mouse model Milestones:--clemizole's in vivo human PK and metabolite profile established --critical drug-drug interactions of clemizole with other key hepatitis C drugs determined. Aim 2-Analyze in vivo resistance and in vivo synergy with PIs by: a) determining resistance mutations in clemizole treated humanized mice infected with HCV b) sequencing and analysis of patient-derived samples for candidate resistant mutations c) determining in vivo synergy of clemizole with advanced PIs in humanized mouse model Milestones:--candidate resistance profile established that can help focus resistance monitoring in patients. --In vivo synergy potential of clemizole with key HCV protease inhibitors established. Aim 3-Complete essential remaining preclinical components needed for successful submission of a US IND, and further advancement to successful commercialization by performing: a) Core battery of safety testing and screening meeting modern testing requirements and methods b) Single dose escalation toxicology studies and multiple dose escalation 90 day rodent and dog studies using current requirements and methods Milestones:-- complete preclinical toxicity studies to modern standards required for filing an IND. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) remains an important cause of worldwide liver disease for which current therapies are inadequate. We have discovered a novel target within the HCV NS4B protein and identified a small molecule- the first generation antihistamine clemizole--as a potent inhibitor of this target. Because clemizole was widely used in the 1950s and 60s with remarkable safety and tolerability, and shows great promise in vitro and in vivo against HCV, we propose to further study this compound's in vivo synergy potential with other anti-HCV agents, resistance profile, major metabolites, drug-drug interactions with other anti-HCV agents, and to update clemizole's pre-clinical package with current safety standards and methods so as to enable an IND submission, in the hope of developing an exciting new treatment option for patients.